The proposed research concerns the in vitro evolution of nucleic acid enzymes with desirable functional properties. The goal of this research is two-fold: first, to develop novel RNA and DNA enzymes that will have applications in biosynthesis and biomedicine;and second, to employ in vitro evolution as a means to understand the processes of Darwinian evolution at the molecularlevel.These goals will be addressed by five research projects that extend work carried out during the previous project period and that seek to develop new approaches for the directed evolution of functional macromolecules. Two of the projects aim to devise novel amplification methods that will have applications inmolecular diagnostics and biosensing. One of these involves a DNA-catalyzedversion of the ligase chain reaction, which could be used to amplify specific DNA sequences, while avoiding some of the limitationsof current protein-catalyzed methods. The other concerns ligand-dependent exponential amplification as a means for detecting particular small molecules or proteins. A third project has potential applications in biosynthesis. It involves the in vitro evolution of nucleic acid enzymes with aldolase activity, especiallywith the abilityto catalyze crossed aldol reactions in a substrate-specific manner. The remaining two projects will explore new approaches for the directed evolution of functional macromolecules.One of these involves a novel microfluidic-based system for the continuous in vitro evolution of ribozymes. It will be used to address the question of how an evolving population escapes from a deep local fitness optimum, and the relationship between mutation frequency and selection pressure during the course of evolutionary optimization. The final project concerns nanostructured biomaterials.It aims to confer precise three-dimensional positioning to functional nucleic acidsby anchoring them to a rigid macromolecular scaffold, composed of a single- stranded DNA that folds into the shape of a regular octahedron. Each of the 12 struts of the DNA octahedron has a different sequence, which will be used to position functional DNAs at specific locations. These decorated nanostructures then will be subject to in vitro evolution, selecting on the basis of both their form and the function of the appended DNAs.